In order to propel the requisite cardiac output through the lungs, the right ventricle must develop a pulmonary arterial pressure sufficient to overcome the resistance to blood flow offered by the pulmonary vasculature. According to the equation, pulmonary arterial pressure=cardiac output pulmonary vascular resistance, the blood pressure within the pulmonary circulation must increase (pulmonary hypertension must develop) whenever the cardiac output cannot be accommodated by the pulmonary vascular capacity. In this context, the vascular capacity is broadly defined to encompass anatomical constraints related to the compliance and volume of the blood vessels, as well as metabolic limitations related to the tone (state of contracture) maintained by the resistance vessels (Wideman and Bottje, 1993; Wideman et al, 1996a,b, 1998a,b, 1999a,b). In broiler chickens, pulmonary hypertension initiates a distinctive pathophysiological progression that terminates as pulmonary hypertension syndrome, also commonly known as ascites (Julian, 1993, Odom, 1993; Wideman and Bottje, 1993; Wideman, 1999). Broilers exhibit a similar pathophysiological progression under a variety of environmental and management conditions, providing support for the hypothesis that increases in the cardiac output and an inadequate pulmonary vascular capacity constitute common mechanisms through which multiple factors can initiate pulmonary hypertension leading to ascites. For example, fast growth and cool temperatures elevate the cardiac output and serve as the most common triggers for ascites in broilers reared near sea level, whereas hypoxic pulmonary vasoconstriction contributes to high incidences of ascites in broilers reared at high altitudes (Cueva et al., 1974; Huchzermeyer and DeRuyck, 1986; Julian, 1993; Odom, 1993; Wideman and Bottje, 1993; Roush et al., 1996, 1997; Wideman, 1997; Wideman et al., 1996a,b, 1998a,b,c, 1999a,b).
A genetic component of ascites susceptibility has been revealed by surgically occluding one pulmonary artery in male and female broiler breeder parents. Unilateral pulmonary artery occlusion applies extremely rigorous and directly focused selection pressure, causing those individuals incapable of tolerating a direct doubling of the pulmonary vascular resistance to rapidly develop ascites. The survivors of chronic unilateral pulmonary artery occlusion apparently possess a cardio-pulmonary capacity sufficient to accommodate the combined challenges of an elevated pulmonary vascular resistance, a disproportionately high rate of blood flow through the unoccluded lung, and sustained pulmonary hypertension (Wideman and Kirby, 1995, 1996; Wideman et al., 1996a,b, 1997). First generation broiler breeder survivors of unilateral pulmonary artery occlusion, subsequently produced male and female progeny exhibiting about a 50% reduction in ascites susceptibility when grown as rapidly as possible during exposure to cool temperatures (Wideman and French, 1999a). Survivors of a second generation of selection, subsequently produced progeny exhibiting about a 90% reduction in ascites susceptibility when compared with the base population from which the ascites resistant line was developed (Wideman and French, 1999b). This rapid pace of genetic improvement confirms the central contribution of an inadequate pulmonary vascular capacity to the ascites susceptibility of broilers. Furthermore, the gene or genes involved in ascites susceptibility appear to be dominant, indicating ongoing proactive exposure and elimination of susceptible individuals will be required to achieve an overall improvement in ascites resistance (Wideman and French, 1999a,b).
The unilateral pulmonary artery occlusion technique is impractical for large-scale genetic selection programs, because considerable time and surgical expertise are required to correctly clamp the delicate and poorly accessible pulmonary artery. More efficient methodologies for triggering controlled, sustainable increases in pulmonary vascular resistance are needed before ascites susceptibility can be eliminated routinely from commercial broiler populations. Chemical mediators of pulmonary vasoconstriction are expensive, and tend to produce transient responses unsuitable for maintaining the pulmonary hypertension necessary to expose ascites susceptibility (Wideman et al., 1998a, 1999a; Wideman, 1999).
This invention relates to a new process for inducing pulmonary hypertension in animals, with an objective of identifying and/or eliminating susceptible individuals or families, as well as hypertension resistant ones to achieve genetic improvement in agriculturally important breeds of poultry (broiler chickens and turkeys), and cattle. The invention also will be useful for animal research directed toward understanding pulmonary hypertension and its sequelae in human patients as well as in animals. Sustained pulmonary hypertension leads to pulmonary hypertension syndrome which adversely impacts poultry production throughout the world, as well as cattle production when cattle are kept at altitudes sufficiently high to challenge blood oxygenation. In poultry, the pathophysiological progression of pulmonary hypertension syndrome leads to terminal ascites (fluid accumulation in the abdominal cavity) followed by death of the animal.
A basis of the invention is the novel concept that particulate substances of a size (approximately 8 to 250 xcexcm diameter) suitable for occluding blood vessels in the lungs (pulmonary vasculature) can be suspended in an appropriate carrier vehicle, and the suspension then can be injected intravenously. The venous blood then carries the particles to the right ventricle of the heart, which in turn pumps the blood containing the particles to the lungs. The particles directly increase pulmonary vascular resistance by lodging in small blood vessels, thereby partially blocking blood blow. Proportional or variable occlusion of the pulmonary vasculature can be accomplished by adjusting the number of particles administered. By increasing the resistance to pulmonary blood flow, the right ventricle of the heart is forced to develop and maintain an elevated pressure in the pulmonary arteries (pulmonary hypertension) to propel the requisite cardiac output through the vessels remaining unoccluded. Animals that are susceptible to pulmonary hypertension subsequently will develop pulmonary hypertension syndrome. Animals that are resistant to pulmonary hypertension will not develop pulmonary hypertension syndrome. The resistant animals or their pedigreed families can be selected for breeding genetic stocks that are resistant to the onset of pulmonary hypertension, pulmonary hypertension syndrome, and/or ascites. Increasing or decreasing the numbers of particles administered can be used to increase or decrease, respectively, the degree of pulmonary vascular obstruction and thereby the selection pressure applied to the animal population. Researchers also may use this as an experimental technique for triggering pulmonary hypertension in experimental animals. This invention also may trigger a pulmonary vascular inflammatory response, which will be useful in research directed toward understanding the responses of the immune system within the lungs.
This invention has the following advantages: (a) individual animals can be evaluated and/or selected based on simple, inexpensive intravenous injections; (b) relatively unskilled personnel can treat thousands of animals per day; (c) the degree of selection pressure can be varied; (d) before and after the injections, the animals can be maintained in typical production facilities without additional expensive requirements for inducing pulmonary hypertension; and, (e) the invention directly/physically affects the pulmonary vasculature without requiring ongoing pharmacological intervention.
Present genetic selection techniques involve prolonged exposure to cool temperatures or hypobaric hypoxia (low atmospheric pressure and thus low oxygen), coupled with various measurements of symptoms related to the onset of pulmonary hypertension and the progression to pulmonary hypertension syndrome (see Wideman et al., 1998. Poultry Science 77:1565-1573). These Conditions require specialized, expensive facilities that generally can accommodate relatively few individuals, and that generally do not apply sufficient selection pressure to achieve rapid genetic progress.
Alternatively, surgical occlusion of one pulmonary artery has been used to trigger a high incidence of pulmonary hypertension syndrome in broiler chickens. The resistant survivors are known to produce progeny that are highly resistant to pulmonary hypertension syndrome (see Wideman et al., 1997. Poultry Science 76:400-404; Wideman and French, 1999. Poultry Science 78:404-411; Wideman and French, 1999). This research proved the validity of partially obstructing the pulmonary vasculature as a means for selecting genetically resistant individuals. However, sophisticated surgical skills and equipment are required for unilateral pulmonary artery occlusion, only 100 birds can be prepared by a two-person team per day, and the process is quite expensive.
The present invention involves an animal exhibiting pulmonary hypertension syndrome, the animal being produced by the administration of pulmonary vasculature-blocking particles to the pulmonary vasculature.
Also involved is a process for producing a population of animals having increased resistance to pulmonary hypertension, the process comprising eliminating animals most susceptible to pulmonary hypertension induced by intravascular particle administration.
This invention may also be used to achieve genetic improvements and other agriculturally important breeds. This is particularly true for breeds subject to pulmonary hypertension and its sequelae. In another aspect, this invention involves for producing improved genetic breeding stocks. This again involves inducing pulmonary hypertension in animals, the method comprising: a) inducing pulmonary hypertension in animals by occluding the pulmonary vasculature with particulate substances of a size about 8 to about 250 micrometers in diameter injected into veneous blood that carries the particles to the right cardiac ventricle pumping the blood containing the particles to the lungs, wherein the particles directly increase pulmonary vascular resistance by lodging in small blood vessels, the heart being forced to develop and maintain an elevated pressure in the pulmonary arteries to propel the requisite cardiac output through vessels remaining unoccluded, animals that are susceptible to pulmonary hypertension subsequently developing pulmonary hypertension syndrome, and b) separating susceptible animals from the resistant animals, the resistant animals being useful as genetic breeding stock resistant to pulmonary hypertension.
Cattle, chickens, pigs and other domestic stock may be subject to pulmonary hypertension, and thus, be the animals of the present invention. In certain cases, the breeding stock produced by the methods of the present invention may be utilized to characterize any novel DNA allelic markers that appear in animals resistant to pulmonary hypertension. Likewise, animals that are killed or sickened by the particle injection of the present invention may be analyzed for genetic allelic DNA markers that are found in such susceptible stock. Those of skill in the art will understand how to do allelic analyses between resistant and susceptible animals and base stock to find the appropriate allelic DNA markers. Once these genetic markers are found, they may be used to select breeding stock.
In summary, the present invention involves a method for producing a pulmonary hypertension resistant poultry strain in its most preferred embodiment. This method involves a) administering into the pulmonary circulation of immature poultry particles having about 8 to about 250 xcexcM diameters in an amount sufficient to kill or disable animals susceptible to pulmonary hypertension; and b) selecting and breeding healthy survivors to produce a pulmonary hypertension resistant poultry strain.